INFECTION AND IMMUNITY, Aug. 1977, p. 425-429 Copyright © 1977 American Society for Microbiology
Vol. 17, No. 2
Printed in U.S.A.
Preparative Polyacrylamide Gel Electrophoresis Purification of Clostridium perfringens Enterotoxin GEORGE L. ENDERS, JR.1, AND CHARLES L. DUNCAN2* Food Research Institute and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 23 February 1977
Preparative polyacrylamide gel electrophoresis has been used to purify the enterotoxin of Clostridium perfringens from Sephadex G-100 extracts. Purified toxin of high specific activity was eluted in 1 to 3 h, depending upon the length of the acrylamide gel used. Recovery of biological activity with this technique ranged from 80 to 90%. The purity and physical characteristics of the toxin were similar to those previously reported for the protein purified by other methods. Use of preparative electrophoresis will enable the production of larger amounts of high-specific-activity toxin in a shorter time than other currently available procedures. This method was also used to isolate a form of enterotoxin that has a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels. culture was then heat-shocked at 75°C for 20 min prior to incubation at 37°C for 16 h. The fluid thioglycolate culture was then transferred (2%, vol/vol) into Duncan and Strong sporulation medium (5, 25) and incubated at 37°C for 7 to 8 h. After harvesting at 4°C, the cells were disrupted by sonic treatment, and debris was removed by centrifugation at 12,000 x g for 20 min at 4°C. The resultant cell extract was used for further purification of the enterotoxin. Sephadex G-100 chromatography. Sephadex G100 gel was swollen in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 7.6) containing 0.1 M KCl and 0.02% sodium azide and poured into a column (2.6 by 70 cm) as previously described (23). Approximately 80 to 100 mg of protein was loaded for each column run, and elution was accomplished with the same buffer. From several column runs, fractions containing toxin, as confirmed by immunodiffusion assay, were pooled, dialyzed, and concentrated as previously described (24). Preparative polyacrylamide gel electrophoresis. Preparative electrophoresis was done in a Buchler Poly Prep 200 apparatus. The cooling jacket was maintained at less than 10°C during polymerization of the gel and throughout the electrophoresis run. The polyacrylamide gel (7% acrylamide) was prepared by the method of Davis (2). A gel 5 cm long was usually prepared from 100 ml of acrylamide solution. Gels prepared as short as 2.5 cm in length proved equally satisfactory and had decreased running times in order to elute the desired protein MATERIALS AND METHODS band. Upper-run buffer consisted of tris(hydroxyCell growth and extracts. Throughout this study methyl)aminomethane (3.0 g) and glycine (14.4 g); C. perfringens type A strain NCTC 8239 was used. lower buffer was the same but at twice the concenCultures grown in cooked meat medium (Difco) were tration. When filling the lower chamber with buffer, used to inoculate fluid thioglycolate medium. The extreme care was taken to insure the removal of all 1 Present address: Miles Laboratories, Ames Division, air bubbles that could interfere with electrophoresis. After polymerization of the donut-shaped gel Elkhart, IN 46414. (about 1 h), the surface of the gel was rinsed with 2 Present address: Campbell Institute for Food Research, upper buffer before filling the upper chamber with Camden, NJ 08101. 425
The enterotoxin elaborated by Clostridium perfringens type A has been demonstrated to be the etiological agent of C. perfringens food poisoning in humans (3, 14-16, 18). The enterotoxin is produced only during sporulation of the cells (8) and is released upon sporangial lysis (4). This toxin has been shown to produce diarrhea in rabbits (6), lambs (13), monkeys (7, 16), and humans (26), fluid accumulation in ligated intestinal loops of lambs (14), rabbits (6), and chickens (21), and erythemal activity in the skin of rabbits and guinea pigs (10, 24). To date, several laboratories have purified this protein by a variety of methods (11, 12, 22, 25). Enterotoxin is usually purified in our laboratory by the method of Stark and Duncan (25), which involves chromatography on Sephadex G-100, Cellex T anion exchange, and hydroxylapatite columns. Recently an affinity chromatography method for purifying the toxin was reported (23). This method is rapid but yields toxin with a much lower specific activity than that reported by Stark and Duncan (25). In this paper we describe a rapid preparative polyacrylamide gel electrophoresis method for the purification of the enterotoxin from C. perfringens type A extracts.
426
INEFECT . IMMUN .
ENDERS AND DUNCAN
buffer for electrophoresis. After further insuring that all of the air bubbles were removed from the elution chamber and that the elution buffer was flowing freely up the elution capillary, the sample was applied to the gel. Bromophenol blue tracking dye was added to the sample (25 to 75 mg) solution to give a faint blue tint. Sixty percent sucrose was added to a final concentration of about 10 to 15% sucrose. This facilitated layering of the sample on the gel. Electrophoresis was conducted at a constant current of 100 mA for the first 30 min and then continued at 175 mA for the remainder of the run. After the tracking dye had completely entered the top of the gel, the upper and lower buffers were both pumped at 1.25 ml/min, and the elution buffer was pumped at the rate of 0.45 ml/min. Approximately 2ml fractions were collected, and the toxin-containing fractions determined by their positive immunodiffusion reactions. Positive fractions were then pooled, dialyzed, and concentrated for further studies. Analytical polyacrylamide gel electrophoresis. Analytical gels were prepared with the same reagents used for the preparative gels. Both electrophoresis buffers had the same concentration as the preparative upper electrophoresis buffer. Gels were electrophoresed at 3 mA/gel until the bromophenol blue tracking dye was about 1 cm from the end of the gel tube. After electrophoresis, the gels were fixed in 12.5% trichloroacetic acid and then stained for 60 min with 0.1% Coomassie brilliant blue G250 in 12.5% trichloroacetic acid. Destaining was accomplished with 7% acetic acid. Densitometer scans were obtained with a Zeineh Soft Laser densitometer (Biomed Instruments, Inc.). Protein determination. Proteins were quantitated by the Lowry et al. method with crystallized bovine serum albumin (Sigma Chemical Co.) as the standard (19). Isoelectric focusing. Gel isoelectric focusing was performed with 6% acrylamide gels according to the MRA Corporation Bulletin no. M137-A-71. Ampholines (pH 3 to 6) were purchased from LKB Producter. Proteins were focused in the gels at 1 mA/tube until the voltage reached 400 V. During the remaining 5 h of the run, the voltage was held constant at 400 V. Focused gels were stained by the methods of Malik and Berrie (20). The pH gradient was determined by slicing a blank gel into 0.5-cm sections and eluting these for about 4 h in a capped vial containing 0.5 ml of freshly boiled distilled water. Immunodiffusion was done in an agar layer consisting of a solution of 1% Noble agar (Difco), 1% NaCl, and 0.01% Merthiolate. Antiserum used for immunodiffusion was prepared against purified enterotoxin from C. perfringens type A as reported by Stark and Duncan (24). Biological activity. Biological activity of the enterotoxin was determined by measurement of the size of the erythema produced when suitable dilutions of toxin were injected intradermally into depilated guinea pigs as previously described (24).
RESULTS AND DISCUSSION
Figure 1 illustrates an elution profile ob-
tained from a 7% preparative polyacrylamide gel loaded with enterotoxin partially purified on Sephadex G-100. A continuous broad absorbancy pattern results, which, as described by Chrambach and Rodbard (1), is often characteristic of preparative polyacrylamide gel profiles. This apparently occurs as a result of high dilution associated with continuous elution and because of overlapping distribution curves of the protein components. Immunodiffusion assay revealed positive precipitin reactions for fractions 19 through 25 and weakly positive reactions for fractions 16 through 18 and 26 through 29. Fractions were numbered from the first tube containing tracking dye, which usually eluted in one or two fractions. Neither fractions (about 10) collected before elution of the dye nor those collected up to 4 h after the toxin had eluted revealed any serological activity with anti-enterotoxin serum. Analytical 7% polyacrylamide gels, with a stacking gel, were used to ascertain the purity of the serologically positive fractions before they were pooled. Figure 2 shows a gel of a pooled, dialyzed, and concentrated sample comprised of fractions 20 through 26 (Fig. 1). Earlier and later serologically active fractions contained contaminating proteins. Some electrophoretic heterogeneity of the toxin is evident from the faint protein band immediately cathodal to the main toxin band. This has been shown previously with purified enterotoxin (23). An additional faint band at the cathode end of the gel did not occur consistently with the purified toxin and may represent aggregated toxin or contaminating protein. It did not react serologically with anti-enterotoxin, but neither does aggregated toxin. The specific ac-
0.4
0.3 E
0.2
-
I
0
10
30 20 Frac t ion Number
40
FIG. 1. Enterotoxin elution profile from a 7% preparative acrylamide gel (5 cm long). The gel was loaded with 26.1 mg of protein previously fractionated on a Sephadex G-100 column. Serologically positive enterotoxin-containing fractions are identified. Details of electrophoresis are given in the text.
VOL. 17, 1977
PREPARATIVE ELECTROPHORESIS OF ENTEROTOXIN
427
FIG. 2. Analytical gels (7%) of Sephadex G-100 fractionated enterotoxin (A) and preparative gel electro-
phoresis purified toxin from fractions 20 through 26 (B). Migration is toward the anode.
tivity and recovery from this preparative electrophoretic run are illustrated in Table 1. Enterotoxin purified by this method showed the same properties (molecular weight, mobility in gels, isoelectric point) as previously reported (24, 25). With this method, the recovery of biological activity ranged from 80 to 91%, with specific activity values ranging from 4,500 to 6,400 erythemal units per mg of protein. Purified toxin has been obtained from preparative gels loaded with 15 to 75 mg of protein, but highest purity was obtained when less than 50 mg was loaded. Elution of the toxin was usually accomplished within 2 to 3 h after initiation of electrophoresis for a 5-cm-long gel, or in a proportionately reduced time for a shorter gel. Comparable recoveries and specific toxin activities were obtained with this technique as compared with the chromatographic procedure of Stark and Duncan (25), but the time required was significantly reduced. In comparison to affinity chromatography (23), the preparative method produces greater amounts (due to the increased loading capacity of the gel) of higher-specific-activity
toxin in a similar time period. The preparative electrophoresis was not satisfactory for purifying enterotoxin from crude cell extracts. This method has also been useful in isolating a fast-migrating protein band that has been observed in some toxin preparations in our laboratory. This protein component reacts serologically with anti-enterotoxin serum. It exhibited a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels, and eluted in one or two fractions about 25 min after elution of the tracking dye. The immunodiffusion slides had to be examined carefully to avoid missing the faint precipitin reaction in this fraction. This protein also produced a weak erythemal reaction that could be neutralized by anti-enterotoxin serum. Ferguson plots (9, 17) (Fig. 3A) indicate that the toxin and the fastmigrating toxin band differ significantly in their charge. Although the weight of the highly charged protein is not significantly greater than the toxin (Fig. 3B), one might speculate that a small, highly charged fragment is lost from the fast-migrating toxin, which results in decreased mobility of the purified toxin. Thus,
428
INF ECT . IMMUN .
ENDERS AND DUNCAN
TABLE 1. Enterotoxin recovery from preparative electrophoresis of Sephadex G-100 fractionated enterotoxin Biological activity Sp act Sample
Protein (mg)
EU
26.1 2.0 4.8
G-100 fractionated protein Serologically positive fractions contaminated with other proteins Serologically positive enterotoxin fractions not contaminated with other proteins (fractions 20 through 26) a EU, Erythemal units. 1.0
%
EU/mg
EU/mg
Fold purifica-
38,288 3,964
100 10.4
1,467 1,982
1.0 1.4
30,820
80.5
6,421
4.4
EUa
I_fA A >,{est migreting
to.in
~~~~~~~~tion
20
sIope=-0.056 0.6
0
-4 0.6 inti 0. slope l.a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
.so .
*
0.2
4~~~~~~~~~~~~~~
0
0
2
4
Percent
6
a
10
12
2
Acrytamide
0
0.2
0.4
0.6
068
1.0
10 1S1.p
FIG. 3. Determination of size and charge differences between the toxin and the fast-migrating toxin component. (A) Influence of acrylamide concentration on the mobility of these components. (B) Molecular weight determinations of components, using the slopes calculated from a plot of the log of relative mobility versus the acrylamide concentration as shown in (A). Protein standards are (1) myoglobin, (2) deoxyribonuclease, and (3-5) bovine serum albumin monomer, dimer, and trimer, respectively.
the toxin could be synthesized as a larger molecule, perhaps a prototoxic form, and cleavage at a later time could be an activation step. Alternatively, it is possible that a gross conformation change could result in more highly charged groups residing on the exterior surface of the protein. Additional work on this aspect will depend on the accumulation of sufficient amounts of this protein component for further characterization. ACKNOWLEDGMENTS This research was supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, by Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases, by Public Health Service research grant FD-00203-07 from the Food and Drug Administration, and by contributions to the Food Research Institute by member industries. G.L.E. is the recipient of a postdoctoral award from Public Health Service grant T32-EF0715-01 from the National Institute of Environmental Health Sciences. C.L.D. is the recipient of a Public Health Service Research Career Development Award AI-70721-03 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Chrambach, A., and D. Rodbard. 1971. Polyacrylamide gel electrophoresis. Science 172:440-451. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. 3. Dische, F. E., and S. D. Elek. 1957. Experimental food poisoning by Clostridium welchii. Lancet ii:71-74. 4. Duncan, C. L. 1973. Time of enterotoxin formation and release during sporulation of Clostridium perfringens type A. J. Bacteriol. 113:932-936. 5. Duncan, C. L., and D. H. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 6. Duncan, C. L., and D. H. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94. 7. Duncan, C. L., and D. H. Strong. 1971. Clostridium perfringens type A food poisoning. I. Response of the rabbit ileum as an indication of enteropathogenicity of strains of Clostridium perfringens in monkeys. Infect. Immun. 3:167-170. 8. Duncan, C. L., D. H. Strong, and M. Sebald. 1972. Sporulation and enterotoxin production by mutants of Clostridium perfringens. J. Bacteriol. 110:378-391. 9. Ferguson, K. A. 1964. Starch gel electrophoresis-ap-
VOL. 17, 1977 10. 11.
12.
13.
14.
15. 16.
17.
PREPARATIVE ELECTROPHORESIS OF ENTEROTOXIN
plication to the classification ofpituitary proteins and polypeptides. Metabolism 13:985-1002. Hauschild, A. H. W. 1970. Erythemal activity of the cellular enteropathogenic factor of Clostridium perfringens type A. Can. J. Microbiol. 16:651-654. Hauschild, A. H. W., and R. Hilsheimer. 1971. Purification and characteristics ofthe enterotoxin of Clostridium perfringens type A. Can. J. Microbiol. 17:14251433. Hauschild, A. H. W., R. Hilsheimer, and W. G. Martin. 1973. Improved purification and further characterization of Clostridium perfringens type A enterotoxin. Can. J. Microbiol. 19:1379-1382. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1970. Enteropathogenic factors of food-poisoning Clostridium perfringens type A. Can. J. Microbiol. 16:331-338. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1971. The role of enterotoxin in Clostridium perfringens type A enteritis. Can. J. Microbiol. 17:987-991. Hauswhild, A. H. W., and F. S. Thatcher. 1967. Experimental food poisoning with heat-susceptible Clostridium perfringens type A. J. Food Sci. 32:467-469. Hauschild, A. H. W., M. J. Walcroft, and W. Campbell. 1971. Emesis and diarrhea induced by enterotoxin of Clostridium perfringens type A in monkeys. Can. J. Microbiol. 17:1141-1143. Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164.
429
18. Hobbs, B. C., S. F. Smith, C. T. Oakley, G. H. Warrack, and J. F. Cruickshank. 1953. Clostridium welchii food poisoning. J. Hyg. 51:74-101. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. Malik, N., and A. Berrie. 1972. New stain fixative for proteins separated by gel isoelectric focusing based on Coomassie Brilliant Blue. Anal. Biochem. 49:173176. 21. Niilo, L. 1974. Response of ligated intestinal loops in chickens to the enterotoxin of Clostridium perfringens. Appl. Microbiol. 28:889-891. 22. Sakaguchi, G., T. Uemera, and H. P. Reiman. 1973. Simplified method for purification of Clostridium perfringens type A enterotoxin. Appl. Microbiol. 26:762767. 23. Scott, V. N., and C. L. Duncan. 1975. Affinity chromatography purification of Clostridium perfringens enterotoxin. Infect. Immun. 12:536-543. 24. Stark, R. L., and C. L. Duncan. 1971. Biological characterization ofClostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 25. Stark, R. L., and C. L. Duncan. 1972. Purification and biochemical properties of Clostridium perfringens type A enterotoxin. Infect. Immun. 6:662-673. 26. Strong, D. H., C. L. Duncan, and G. Perna. 1971. Clostridium perfringens type A food poisoning. II. Response of the rabbit ileum as an indication of enteropathogenicity of strains of Clostridium perfringens in human beings. Infect. Immun. 3:171-178.
Vol. 17, No. 2
Printed in U.S.A.
Preparative Polyacrylamide Gel Electrophoresis Purification of Clostridium perfringens Enterotoxin GEORGE L. ENDERS, JR.1, AND CHARLES L. DUNCAN2* Food Research Institute and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 23 February 1977
Preparative polyacrylamide gel electrophoresis has been used to purify the enterotoxin of Clostridium perfringens from Sephadex G-100 extracts. Purified toxin of high specific activity was eluted in 1 to 3 h, depending upon the length of the acrylamide gel used. Recovery of biological activity with this technique ranged from 80 to 90%. The purity and physical characteristics of the toxin were similar to those previously reported for the protein purified by other methods. Use of preparative electrophoresis will enable the production of larger amounts of high-specific-activity toxin in a shorter time than other currently available procedures. This method was also used to isolate a form of enterotoxin that has a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels. culture was then heat-shocked at 75°C for 20 min prior to incubation at 37°C for 16 h. The fluid thioglycolate culture was then transferred (2%, vol/vol) into Duncan and Strong sporulation medium (5, 25) and incubated at 37°C for 7 to 8 h. After harvesting at 4°C, the cells were disrupted by sonic treatment, and debris was removed by centrifugation at 12,000 x g for 20 min at 4°C. The resultant cell extract was used for further purification of the enterotoxin. Sephadex G-100 chromatography. Sephadex G100 gel was swollen in 0.05 M tris(hydroxymethyl)aminomethane buffer (pH 7.6) containing 0.1 M KCl and 0.02% sodium azide and poured into a column (2.6 by 70 cm) as previously described (23). Approximately 80 to 100 mg of protein was loaded for each column run, and elution was accomplished with the same buffer. From several column runs, fractions containing toxin, as confirmed by immunodiffusion assay, were pooled, dialyzed, and concentrated as previously described (24). Preparative polyacrylamide gel electrophoresis. Preparative electrophoresis was done in a Buchler Poly Prep 200 apparatus. The cooling jacket was maintained at less than 10°C during polymerization of the gel and throughout the electrophoresis run. The polyacrylamide gel (7% acrylamide) was prepared by the method of Davis (2). A gel 5 cm long was usually prepared from 100 ml of acrylamide solution. Gels prepared as short as 2.5 cm in length proved equally satisfactory and had decreased running times in order to elute the desired protein MATERIALS AND METHODS band. Upper-run buffer consisted of tris(hydroxyCell growth and extracts. Throughout this study methyl)aminomethane (3.0 g) and glycine (14.4 g); C. perfringens type A strain NCTC 8239 was used. lower buffer was the same but at twice the concenCultures grown in cooked meat medium (Difco) were tration. When filling the lower chamber with buffer, used to inoculate fluid thioglycolate medium. The extreme care was taken to insure the removal of all 1 Present address: Miles Laboratories, Ames Division, air bubbles that could interfere with electrophoresis. After polymerization of the donut-shaped gel Elkhart, IN 46414. (about 1 h), the surface of the gel was rinsed with 2 Present address: Campbell Institute for Food Research, upper buffer before filling the upper chamber with Camden, NJ 08101. 425
The enterotoxin elaborated by Clostridium perfringens type A has been demonstrated to be the etiological agent of C. perfringens food poisoning in humans (3, 14-16, 18). The enterotoxin is produced only during sporulation of the cells (8) and is released upon sporangial lysis (4). This toxin has been shown to produce diarrhea in rabbits (6), lambs (13), monkeys (7, 16), and humans (26), fluid accumulation in ligated intestinal loops of lambs (14), rabbits (6), and chickens (21), and erythemal activity in the skin of rabbits and guinea pigs (10, 24). To date, several laboratories have purified this protein by a variety of methods (11, 12, 22, 25). Enterotoxin is usually purified in our laboratory by the method of Stark and Duncan (25), which involves chromatography on Sephadex G-100, Cellex T anion exchange, and hydroxylapatite columns. Recently an affinity chromatography method for purifying the toxin was reported (23). This method is rapid but yields toxin with a much lower specific activity than that reported by Stark and Duncan (25). In this paper we describe a rapid preparative polyacrylamide gel electrophoresis method for the purification of the enterotoxin from C. perfringens type A extracts.
426
INEFECT . IMMUN .
ENDERS AND DUNCAN
buffer for electrophoresis. After further insuring that all of the air bubbles were removed from the elution chamber and that the elution buffer was flowing freely up the elution capillary, the sample was applied to the gel. Bromophenol blue tracking dye was added to the sample (25 to 75 mg) solution to give a faint blue tint. Sixty percent sucrose was added to a final concentration of about 10 to 15% sucrose. This facilitated layering of the sample on the gel. Electrophoresis was conducted at a constant current of 100 mA for the first 30 min and then continued at 175 mA for the remainder of the run. After the tracking dye had completely entered the top of the gel, the upper and lower buffers were both pumped at 1.25 ml/min, and the elution buffer was pumped at the rate of 0.45 ml/min. Approximately 2ml fractions were collected, and the toxin-containing fractions determined by their positive immunodiffusion reactions. Positive fractions were then pooled, dialyzed, and concentrated for further studies. Analytical polyacrylamide gel electrophoresis. Analytical gels were prepared with the same reagents used for the preparative gels. Both electrophoresis buffers had the same concentration as the preparative upper electrophoresis buffer. Gels were electrophoresed at 3 mA/gel until the bromophenol blue tracking dye was about 1 cm from the end of the gel tube. After electrophoresis, the gels were fixed in 12.5% trichloroacetic acid and then stained for 60 min with 0.1% Coomassie brilliant blue G250 in 12.5% trichloroacetic acid. Destaining was accomplished with 7% acetic acid. Densitometer scans were obtained with a Zeineh Soft Laser densitometer (Biomed Instruments, Inc.). Protein determination. Proteins were quantitated by the Lowry et al. method with crystallized bovine serum albumin (Sigma Chemical Co.) as the standard (19). Isoelectric focusing. Gel isoelectric focusing was performed with 6% acrylamide gels according to the MRA Corporation Bulletin no. M137-A-71. Ampholines (pH 3 to 6) were purchased from LKB Producter. Proteins were focused in the gels at 1 mA/tube until the voltage reached 400 V. During the remaining 5 h of the run, the voltage was held constant at 400 V. Focused gels were stained by the methods of Malik and Berrie (20). The pH gradient was determined by slicing a blank gel into 0.5-cm sections and eluting these for about 4 h in a capped vial containing 0.5 ml of freshly boiled distilled water. Immunodiffusion was done in an agar layer consisting of a solution of 1% Noble agar (Difco), 1% NaCl, and 0.01% Merthiolate. Antiserum used for immunodiffusion was prepared against purified enterotoxin from C. perfringens type A as reported by Stark and Duncan (24). Biological activity. Biological activity of the enterotoxin was determined by measurement of the size of the erythema produced when suitable dilutions of toxin were injected intradermally into depilated guinea pigs as previously described (24).
RESULTS AND DISCUSSION
Figure 1 illustrates an elution profile ob-
tained from a 7% preparative polyacrylamide gel loaded with enterotoxin partially purified on Sephadex G-100. A continuous broad absorbancy pattern results, which, as described by Chrambach and Rodbard (1), is often characteristic of preparative polyacrylamide gel profiles. This apparently occurs as a result of high dilution associated with continuous elution and because of overlapping distribution curves of the protein components. Immunodiffusion assay revealed positive precipitin reactions for fractions 19 through 25 and weakly positive reactions for fractions 16 through 18 and 26 through 29. Fractions were numbered from the first tube containing tracking dye, which usually eluted in one or two fractions. Neither fractions (about 10) collected before elution of the dye nor those collected up to 4 h after the toxin had eluted revealed any serological activity with anti-enterotoxin serum. Analytical 7% polyacrylamide gels, with a stacking gel, were used to ascertain the purity of the serologically positive fractions before they were pooled. Figure 2 shows a gel of a pooled, dialyzed, and concentrated sample comprised of fractions 20 through 26 (Fig. 1). Earlier and later serologically active fractions contained contaminating proteins. Some electrophoretic heterogeneity of the toxin is evident from the faint protein band immediately cathodal to the main toxin band. This has been shown previously with purified enterotoxin (23). An additional faint band at the cathode end of the gel did not occur consistently with the purified toxin and may represent aggregated toxin or contaminating protein. It did not react serologically with anti-enterotoxin, but neither does aggregated toxin. The specific ac-
0.4
0.3 E
0.2
-
I
0
10
30 20 Frac t ion Number
40
FIG. 1. Enterotoxin elution profile from a 7% preparative acrylamide gel (5 cm long). The gel was loaded with 26.1 mg of protein previously fractionated on a Sephadex G-100 column. Serologically positive enterotoxin-containing fractions are identified. Details of electrophoresis are given in the text.
VOL. 17, 1977
PREPARATIVE ELECTROPHORESIS OF ENTEROTOXIN
427
FIG. 2. Analytical gels (7%) of Sephadex G-100 fractionated enterotoxin (A) and preparative gel electro-
phoresis purified toxin from fractions 20 through 26 (B). Migration is toward the anode.
tivity and recovery from this preparative electrophoretic run are illustrated in Table 1. Enterotoxin purified by this method showed the same properties (molecular weight, mobility in gels, isoelectric point) as previously reported (24, 25). With this method, the recovery of biological activity ranged from 80 to 91%, with specific activity values ranging from 4,500 to 6,400 erythemal units per mg of protein. Purified toxin has been obtained from preparative gels loaded with 15 to 75 mg of protein, but highest purity was obtained when less than 50 mg was loaded. Elution of the toxin was usually accomplished within 2 to 3 h after initiation of electrophoresis for a 5-cm-long gel, or in a proportionately reduced time for a shorter gel. Comparable recoveries and specific toxin activities were obtained with this technique as compared with the chromatographic procedure of Stark and Duncan (25), but the time required was significantly reduced. In comparison to affinity chromatography (23), the preparative method produces greater amounts (due to the increased loading capacity of the gel) of higher-specific-activity
toxin in a similar time period. The preparative electrophoresis was not satisfactory for purifying enterotoxin from crude cell extracts. This method has also been useful in isolating a fast-migrating protein band that has been observed in some toxin preparations in our laboratory. This protein component reacts serologically with anti-enterotoxin serum. It exhibited a mobility, relative to bromophenol blue tracking dye, of 0.87 to 0.90 in 7% acrylamide gels, and eluted in one or two fractions about 25 min after elution of the tracking dye. The immunodiffusion slides had to be examined carefully to avoid missing the faint precipitin reaction in this fraction. This protein also produced a weak erythemal reaction that could be neutralized by anti-enterotoxin serum. Ferguson plots (9, 17) (Fig. 3A) indicate that the toxin and the fastmigrating toxin band differ significantly in their charge. Although the weight of the highly charged protein is not significantly greater than the toxin (Fig. 3B), one might speculate that a small, highly charged fragment is lost from the fast-migrating toxin, which results in decreased mobility of the purified toxin. Thus,
428
INF ECT . IMMUN .
ENDERS AND DUNCAN
TABLE 1. Enterotoxin recovery from preparative electrophoresis of Sephadex G-100 fractionated enterotoxin Biological activity Sp act Sample
Protein (mg)
EU
26.1 2.0 4.8
G-100 fractionated protein Serologically positive fractions contaminated with other proteins Serologically positive enterotoxin fractions not contaminated with other proteins (fractions 20 through 26) a EU, Erythemal units. 1.0
%
EU/mg
EU/mg
Fold purifica-
38,288 3,964
100 10.4
1,467 1,982
1.0 1.4
30,820
80.5
6,421
4.4
EUa
I_fA A >,{est migreting
to.in
~~~~~~~~tion
20
sIope=-0.056 0.6
0
-4 0.6 inti 0. slope l.a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
.so .
*
0.2
4~~~~~~~~~~~~~~
0
0
2
4
Percent
6
a
10
12
2
Acrytamide
0
0.2
0.4
0.6
068
1.0
10 1S1.p
FIG. 3. Determination of size and charge differences between the toxin and the fast-migrating toxin component. (A) Influence of acrylamide concentration on the mobility of these components. (B) Molecular weight determinations of components, using the slopes calculated from a plot of the log of relative mobility versus the acrylamide concentration as shown in (A). Protein standards are (1) myoglobin, (2) deoxyribonuclease, and (3-5) bovine serum albumin monomer, dimer, and trimer, respectively.
the toxin could be synthesized as a larger molecule, perhaps a prototoxic form, and cleavage at a later time could be an activation step. Alternatively, it is possible that a gross conformation change could result in more highly charged groups residing on the exterior surface of the protein. Additional work on this aspect will depend on the accumulation of sufficient amounts of this protein component for further characterization. ACKNOWLEDGMENTS This research was supported by the College of Agriculture and Life Sciences, University of Wisconsin, Madison, by Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases, by Public Health Service research grant FD-00203-07 from the Food and Drug Administration, and by contributions to the Food Research Institute by member industries. G.L.E. is the recipient of a postdoctoral award from Public Health Service grant T32-EF0715-01 from the National Institute of Environmental Health Sciences. C.L.D. is the recipient of a Public Health Service Research Career Development Award AI-70721-03 from the National Institute of Allergy and Infectious Diseases.
LITERATURE CITED 1. Chrambach, A., and D. Rodbard. 1971. Polyacrylamide gel electrophoresis. Science 172:440-451. 2. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. 3. Dische, F. E., and S. D. Elek. 1957. Experimental food poisoning by Clostridium welchii. Lancet ii:71-74. 4. Duncan, C. L. 1973. Time of enterotoxin formation and release during sporulation of Clostridium perfringens type A. J. Bacteriol. 113:932-936. 5. Duncan, C. L., and D. H. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 6. Duncan, C. L., and D. H. Strong. 1969. Ileal loop fluid accumulation and production of diarrhea in rabbits by cell-free products of Clostridium perfringens. J. Bacteriol. 100:86-94. 7. Duncan, C. L., and D. H. Strong. 1971. Clostridium perfringens type A food poisoning. I. Response of the rabbit ileum as an indication of enteropathogenicity of strains of Clostridium perfringens in monkeys. Infect. Immun. 3:167-170. 8. Duncan, C. L., D. H. Strong, and M. Sebald. 1972. Sporulation and enterotoxin production by mutants of Clostridium perfringens. J. Bacteriol. 110:378-391. 9. Ferguson, K. A. 1964. Starch gel electrophoresis-ap-
VOL. 17, 1977 10. 11.
12.
13.
14.
15. 16.
17.
PREPARATIVE ELECTROPHORESIS OF ENTEROTOXIN
plication to the classification ofpituitary proteins and polypeptides. Metabolism 13:985-1002. Hauschild, A. H. W. 1970. Erythemal activity of the cellular enteropathogenic factor of Clostridium perfringens type A. Can. J. Microbiol. 16:651-654. Hauschild, A. H. W., and R. Hilsheimer. 1971. Purification and characteristics ofthe enterotoxin of Clostridium perfringens type A. Can. J. Microbiol. 17:14251433. Hauschild, A. H. W., R. Hilsheimer, and W. G. Martin. 1973. Improved purification and further characterization of Clostridium perfringens type A enterotoxin. Can. J. Microbiol. 19:1379-1382. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1970. Enteropathogenic factors of food-poisoning Clostridium perfringens type A. Can. J. Microbiol. 16:331-338. Hauschild, A. H. W., L. Niilo, and W. J. Dorward. 1971. The role of enterotoxin in Clostridium perfringens type A enteritis. Can. J. Microbiol. 17:987-991. Hauswhild, A. H. W., and F. S. Thatcher. 1967. Experimental food poisoning with heat-susceptible Clostridium perfringens type A. J. Food Sci. 32:467-469. Hauschild, A. H. W., M. J. Walcroft, and W. Campbell. 1971. Emesis and diarrhea induced by enterotoxin of Clostridium perfringens type A in monkeys. Can. J. Microbiol. 17:1141-1143. Hedrick, J. L., and A. J. Smith. 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155-164.
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18. Hobbs, B. C., S. F. Smith, C. T. Oakley, G. H. Warrack, and J. F. Cruickshank. 1953. Clostridium welchii food poisoning. J. Hyg. 51:74-101. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. Malik, N., and A. Berrie. 1972. New stain fixative for proteins separated by gel isoelectric focusing based on Coomassie Brilliant Blue. Anal. Biochem. 49:173176. 21. Niilo, L. 1974. Response of ligated intestinal loops in chickens to the enterotoxin of Clostridium perfringens. Appl. Microbiol. 28:889-891. 22. Sakaguchi, G., T. Uemera, and H. P. Reiman. 1973. Simplified method for purification of Clostridium perfringens type A enterotoxin. Appl. Microbiol. 26:762767. 23. Scott, V. N., and C. L. Duncan. 1975. Affinity chromatography purification of Clostridium perfringens enterotoxin. Infect. Immun. 12:536-543. 24. Stark, R. L., and C. L. Duncan. 1971. Biological characterization ofClostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 25. Stark, R. L., and C. L. Duncan. 1972. Purification and biochemical properties of Clostridium perfringens type A enterotoxin. Infect. Immun. 6:662-673. 26. Strong, D. H., C. L. Duncan, and G. Perna. 1971. Clostridium perfringens type A food poisoning. II. Response of the rabbit ileum as an indication of enteropathogenicity of strains of Clostridium perfringens in human beings. Infect. Immun. 3:171-178.
